Receptor signaling pathways are the subject of widespread research efforts. A better understanding of these signaling pathways will lead to the design of new and more effective drugs in the treatment of many diseases. Of particular interest are the growth factor and related receptor signaling pathways and their role in cell growth and differentiation. Binding of a particular growth factor to its receptor on the cell plasma membrane can stimulate a wide variety of biochemical responses, including changes in ion fluxes, activation of various kinases, alteration of cell shape, transcription of various genes and modulation of enzymatic activities in cellular metabolism.
Growth factors play a role in embryonic development, cancer, atherosclerosis and the responses of tissues to injury. Growth factors are involved in several normal developmental processes as well as in pathological conditions. Many growth factor receptors are tyrosine kinases whose signalling is dependent upon tyrosine phosphorylation of both the receptor and other molecules. Specific phosphorylated tyrosine residues on these receptors recruit soluble intracellular signaling molecules to the complex upon growth factor stimulation, thus initiating the growth factor signaling cascade. The signal can then proceed through a series of steps to the nucleus and other subcellular locations where the final effects of activation by the extracellular ligand are produced. Recruitment of molecules is often carried out by adapter molecules containing only protein xe2x80x94protein interaction domains with no associated enzymatic activity. By examining the molecules that interact with these adapters, important parts of the signaling mechanism can be discovered, monitored and controlled. One such adapter protein is GRB2, a 24 kDa cytosolic adapter protein containing two SH3 domains flanking an SH2 domain, which is known to be involved in linking many important molecules in signal transduction.
Because disregulation of the cellular processes involved in cell growth can have disastrous effects, it is important to understand and gain control over these processes. This requires identifying the participants in the signaling events that lead to mitogenesis and elucidating their mechanism of function. The identification of these participants is important for a wide range of diagnostic, therapeutic and screening applications. In particular, by knowing the structure of a particular participant in a growth factor activation cascade, one can design compounds which affect that cascade, to either activate an otherwise inactive pathway, or inactivate an overly active pathway. Similarly, having identified a particular participant in a growth factor cascade, one can also identify situations where that cascade is defective, resulting in a particular pathological state. The identification of participants in particular growth factor activation cascades is thus of critical importance for screening compounds that affect these cascades and treating a variety of disorders resulting from anomalies in these cascades, both as therapeutic agents and as model systems for identification of compounds which affect the pathway and thus may be useful as therapeutic agents. The present invention meets these and many other needs.
The present invention generally provides substantially pure polypeptides, comprising an amino acid. sequence that is substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.
The present invention also provides isolated nucleic acid segments, which encode a polypeptide having an amino acid sequence substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2), or biologically active fragments thereof.
Also provided are isolated antibodies that are specifically immunoreactive with a polypeptide having an amino acid sequence substantially homologous to the amino acid sequence shown in FIG. 10 (SEQ ID NO:2) or its biologically active fragments.
In a further aspect, the present invention provides methods of using these polypeptides. In particular, the invention provides a method of determining whether a test compound is an agonist or antagonist of a GRB2/GA5Ptase interaction. The method comprises contacting GRB2 with GA5Ptase (SEQ ID NO:2) under conditions conducive to forming a GRB2/GA5Ptase complex, in the presence and absence of the test compound. The amount of GRB2/GA5Ptase complex formed in the presence and absence of the test compound is then determined. An increase or decrease in the amount of GRB2/GA5Ptase complex formed in the presence of the test compound is indicative that the test compound is an agonist or antagonist of GRB2/GA5Ptase interaction, respectively.
In a related aspect, the present invention provides a method for determining whether a test compound is an agonist or antagonist of an inositol polyphosphate 5-phosphatase activity. The method comprises incubating a mixture of inositol polyphosphate substrate and GA5Ptase, in the presence and absence of the test compound. The mixture is then assayed to determine the amount of GA5Ptase product formed in the presence and absence of the test compound. The amount of product of GA5Ptase activity in the presence of the test compound is compared to the amount of product of GA5Ptase activity in the absence of the test compound. An increase or decrease in the amount of product of GA5Ptase activity in the presence of the test compound is indicative that the test compound is an agonist or antagonist of an inositol polyphosphate 5-phosphatase activity, respectively.
The present invention also provides a method of identifying the presence of GRB2 in a sample. The method comprises incubating the sample with the polypeptide of the invention, and detecting binding between the polypeptide and a portion of the sample. This binding is indicative of the presence of GRB2 in the sample.
Also provided is a method of purifying GRB2 from a mixture of different proteins containing GRB2. The method comprises immobilizing the polypeptide of the invention, on a solid support. The mixture of proteins is then contacted with the solid support under conditions in which the polypeptide binds GRB2. The solid support is washed to remove unbound proteins, and GRB2 is eluted from the solid support.
The present invention also provides kits for practicing these methods.
In a further aspect, the present invention provides a method of treating a patient suffering from a proliferative disorder. The method comprises administering to the patient a therapeutically effective amount of the polypeptide of the invention.
The present invention also provides substantially pure polypeptides that are immunologically cross-reactive with antibodies to the GA5ptase polypeptides and fragments, described herein.